Qa-1/NKG2A信号通路调控γδT细胞IGF-1分泌在创面愈合中的作用及机制研究

The healing of wound is the core problem in the therapy of trauma and burn injury. The γδT cells, a subset of T cell which express a γδTCR and most abandance resided in the epithelial tissue, plays an important role in the process of wound healing. The γδT cells could screte growth factors like insulin-like growth factor and keratinocyte growth factor that regulate the proliferation, migaration and appoptosis of the keratocytes and fibroblasts in the wound base, and affect the wound healing process. Qa-1 is a nonclassical class I MHC molecule with roles in the regulation of both innate and adaptive immunity. The ligand ofQa-1 is NKG2A, which forms an additional and distinct class I receptor, adjacentto CD94 and forms a heterodimer was constitutive expressed on γδT and NK cells. CD94/NKG2A could transduce negative signal through ITIM motif and the SHP-1/2 signaling pathway, and suppress the cell lysis activity of γδT cells. Qa-1 could also downregulate the expression and secretion of INF-γ in γδT and NK cells. In our pilot study, the wound healing time of full thickness dermal wound in Qa-1.knockout transgeneic mice was significantly shortened compared with which in the wild-type controls. And this effect could be reversed if the γδT cells deficiency. This result implied that Qa-1/NKG2A block may affect the function of γδT cells and regulate the wound healing process. Based on these results, we hypothesize that the activation of NKG2A on γδT cells by combination with Qa-1, could recuit and activate SHP-2. The SHP-2 then up-regulate the Ras-MEK-ERK signaling pathway, and inhibit the PI3K-Akt-mTOR signaling pathway. The regulation.of the two signaling pathways could inhibit the expression and scretion of IGF-1 by γδT cells. Blocking the interaction between Qa-1 and CD94/NKG2A on γδT cells may release such suppression effect, which could enhance the scretion of IGF-1 by γδT cells, and finally promote wound healing.

皮肤创面愈合过程中，皮肤中的γδT细胞可通过分泌胰岛素样生长因子1（IGF-1），促进创基细胞的增殖与迁移，抑制其凋亡，对创伤愈合有重要的调节作用。Qa-1属非典型MHC分子，其通过与其受体CD94/NKG2A的结合，对γδT细胞的活化具有显著的抑制作用。根据前期研究结果，我们推测γδT细胞表达的NKG2A与其配体Qa-1结合活化后，可通过激活Ras-MEK-ERK信号通路，同时抑制PI3K-Akt-mTOR信号通路，对其IGF-1的表达产生抑制作用。而阻断Qa-1/NKG2A的结合，或可解除其对IGF-1表达的负调控作用，上调γδT细胞IGF-1的表达及分泌，从而促进创面的愈合。本项目拟在皮肤创面模型中，通过在体和体外研究，探讨Qa-1/NKG2A信号通路调控γδT细胞IGF-1分泌在创面愈合中作用及机制，丰富对 γδT细胞在创面修复中的作用与与机制的认识，为创面治疗提供新的策略和靶点。

γδT 细胞是一类主要分布于皮肤粘膜组织的T细胞亚群，是上皮组织免疫微环境的重要组成部分，并可通过分泌IGF-1促进上皮细胞增殖、迁移从而加速上皮化过程，在创面愈合中具有重要作用。NKG2家族受体是γδT细胞主要的免疫共刺激信号分子，对γδT细胞的生物学行为具有重要的调控作用。前期研究发现，阻断NKG2A与其配体Qa-1的信号转导，对于皮肤γδT细胞IGF-1分泌具有抑制作用。故拟对Qa-1信号通路在创面愈合过程中γδT细胞的作用及可能机制进行探索。. 在本项目中，通过皮肤创面模型证实，Qa-1信号通路阻断可导致创面愈合延迟，且创面边缘皮肤组织中DETC细胞的IGF-1表达量显著降低。同时，Qa-1缺陷组创面炎症反应较对照组明显增强，且其Vγ4+T细胞表达、分泌促炎因子IL-17a的水平显著升高。清除小鼠的创面周围皮肤组织的Vγ4+T细胞后，其创面周围组织中的IL-17a 水平显著下降。而在DETC、Vγ4+T细胞体外共培养实验亦证实，Qa-1 KO组培养液上清中及细胞提取的总蛋白中炎性因子IL-17a、 IL-1β、IL-23均增加，而DETC细胞的IGF-1表达下降。Qa-1 KO组DETC细胞的mTOR信号通路中信号分子Akt与S6K的磷酸化水平无显著改变；而其STAT3磷酸化与核转录因子NF-κb表达显著增强。同时，DETC细胞在受到CD3、IL-15刺激活化后高表达PD-L1，而Vγ4+T活化后表达PD-1受体。在DETC、Vγ4+T细胞共培养体系中阻断PD-1信号通路，则Vγ4+T细胞的IL-17a与IFN-γ等促炎因子的表达明显升高，且转录因子Eomes表达显著上调。. 上述研究结果表明，Qa-1并非直接调控γδT细胞的PI3K-Akt-mTOR或Ras信号通路从而影响其IGF-1分泌，而可能是调控皮肤中γδT细胞的两个亚群，即DETC与Vγ4+T细胞间的相互作用，及IL-17A – IL-1β/IL-23炎性反馈环路，从而调控DETC细胞内的NF-kB信号通路，影响IGF-1表达。PD-1信号通路在Vγ4+T细胞介导IL-17A、IFN-γ炎症因子的表达中可能具有重要的负调作用。. 通过该项目研究发现了创面愈合过程中γδT细胞在介导炎症反应中的可能调控机制，对于未来难愈性创面过度炎症的干预治疗提供了的靶点。